1. Field of the Invention
This invention relates to an apparatus and a methodology to determine the presence and/or concentration of certain metabolites or other substances occurring within a certain medium, such as and without limitation, blood, urine, and saliva. In one embodiment the apparatus and methodology utilizing the principles of the invention is adapted for use as a birth management tool.
2. Background of the Invention
Many types of xe2x80x9chome-basedxe2x80x9d testing apparatuses exist to determine the presence and/or concentration of substances such as metabolites occurring within some medium. While these prior xe2x80x9chome-basedxe2x80x9d testing apparatuses often adequately determine the presence of some of these substances, such as metabolites, they are not capable of determining the concentration or, in many cases, even the presence of such target substances when the concentration of these metabolites or other substances is less than about 10xe2x88x926M. In these cases, relatively complex and costly laboratory tests must be done, usually by one or more trained technicians, in order to ascertain the presence and/or concentration of these substances. There is therefore a great need for a technique (hereinafter the term xe2x80x9ctechniquexe2x80x9d as used in this Application refers to both an apparatus and a methodology) to reduce health care cost by providing a relatively inexpensive and relatively easy to use xe2x80x9chome-basedxe2x80x9d testing apparatus to determine the presence and/or concentration of certain substances or metabolites. Applicant""s invention addresses and fulfills this need. Moreover, there is also a great need for a xe2x80x9creal timexe2x80x9d diagnostic technique that will provide reliable data concerning the presence and/or concentration of certain metabolites and other substances occurring within a certain medium and which will allow an individual to take a prompt action in response to the diagnostic indications. Applicant""s invention provides such a xe2x80x9creal timexe2x80x9d diagnostic indication.
Particularly, Applicant has found that these various needs are particularly acute in the field of birth control and birth management and that Applicant""s invention, in one embodiment, is particularly suited for use in birth control and birth management. That is, it has been deduced form a retrospective study of family planning records that about 90% of all conceptions occur within about a five day period which probably spans the day of the peak concentration of the luteinizing hormone occurring and/or most often sensed within human urine, and more particularly, probably spans from about two days before this peak occurs until about three days after the peak arrives. It is known that during this fertile period, sexual intercourse may lead to pregnancy. Hence, many of the current xe2x80x9chome-basedxe2x80x9d birth management techniques utilize some sort of color changeable paper to notify the users of the pending fertility period by measuring the concentration of the luteinizing hormone. While somewhat useful, these prior techniques provide notification of the impending ovulation no more than thirty-six hours prior to the onset of the fertile period. Since the average longevity of sperm in the vagina is about forty-eight hours, these prior xe2x80x9chome-basedxe2x80x9d tests do not detect the start of the fertility cycle sufficiently early to reliably prevent conception and/or to actually xe2x80x9cmanagexe2x80x9d the conception process.
It is known that the 17-estradiol metabolite, estrone-3-glucuronide (commonly referred to by those of ordinary skill in the art as xe2x80x9cE1-gxe2x80x9d) reaches about 85% to about 95% of its peak value in human urine within about 72 hours prior to the onset of ovulation. Hence, the concentration measurements of E1-g in urine, as Applicant has found, provides a reliable advance warning of the onset of ovulation, sufficient to prevent conception since the time of warning is longer that the lifetime of sperm in the vagina and sufficient to allow individuals to xe2x80x9cmanagexe2x80x9d conception (e.g. actively determine and plan when conception should begin). Prior xe2x80x9chome-basedxe2x80x9d techniques are not readily able to measure E1-g within the urine because the gradual monotonic peak of E1-g requires a more quantitative detection device than current tests provide. Moreover, the World Health Organization has determined the xe2x80x9chome-basedxe2x80x9d E1-g birth management systems would be of great utility in managing the world""s population and would be especially useful in overpopulated and developing countries since birth management could be achieved (e.g. birth control could be achieved by abstinence during the ovulation period while conception could be more readily achieved by sexual intercourse during ovulation). Applicant""s invention, in one embodiment, provides such an E1-g sensing and/or testing apparatus for use in xe2x80x9chome-basedxe2x80x9d birth management and provides the utility sought in the developing and overpopulated countries. Applicant""s invention is therefore an advance in the art of birth management and contains general inventive principles which have a wide use in many other areas of sensing systems.
It is a first objective of the present invention to provide a rapid measurement technique for determining the presence and/or concentration of certain analytes (metabolites and/or other substances within a medium such as urine, blood, and/or saliva) which overcomes the various disadvantages of the prior art.
It is a second object of the present invention to provide a xe2x80x9chome-basedxe2x80x9d technique to determine the presence and/or concentration of certain analytes and other substances within a medium.
It is a third object of the invention to provide a technique to determine the concentration of the analyte, E1-g, in a medium.
It is a fourth object of the present invention to provide a technique which generates a certain color which is indicative of the concentration of a certain analyte, such as E1-g, within a medium.
It is a fifth object of the present invention to provide a technique to provide for xe2x80x9chome-basedxe2x80x9d birth management in a manner which is superior to that of the prior art.
It is a sixth object of the present invention to provide a technique for measuring the concentration and/or occurrence of a certain target analyte occurring within a medium by the use of first and second substances which compete for entry into a membrane which is constrained to remain electrically neutral.
It is a seventh object of the present invention to provide a technique utilizing a matrix type strip which is adapted for placement within a target medium and which is further adapted to provide a certain color indication at a certain portion thereof, indicative of the concentration of a certain target analyte occurring within the target medium.
It is an eighth object of the present invention to provide a technique utilizing a continuous strip which is adapted for placement within a target medium and which is further adapted to display certain color indication whose position along the strip is indicative of the concentration of a certain target analyte occurring within the target medium.
It is a ninth object of the present invention to provide an affinophore molecule which is synthesized so as to have a certain binding affinity for antibody to estrone 3-glucuronide, which allows the biological recognition molecule to modulate the entry of two competing substances within a membrane by an amount which is proportional to the concentration of a certain third substance within the medium. In general, an affinophore for use in Applicant""s techniques is created from a commercially available ionophore which has been chemically and/or biologically altered or conjugated with or to a molecule so as to have a binding affinity for a biological recognition molecule which has a binding affinity for the metabolite or other substance whose concentration is to be measured. The biological recognition molecule may alternatively comprise an antibody, a portion of an antibody, a biological receptor for the analyte, a portion of a nucleotide sequence having chemical and/or biological affinity for the analyte, or virtually any other substance which has some chemical and/or biological binding affinity for the analyte of interest.
According to a first aspect of the present invention, an apparatus to determine the concentration of a certain metabolite occurring within a solution is provided. The apparatus comprises a test strip which changes color in response to the binding of an analyte-specific biological recognition molecule (e.g. an antibody, a receptor, a piece of DNA) or a portion of such a recognition molecule which retains relevant binding characteristics, and its binding partner. The color change results from response of a lipophilized chromophore which changes its color (or fluorescence) in response to the concentration of the specific ion (such as H+). Such a molecule is referred to as a xe2x80x9cchromionophore (or fluorionophore)xe2x80x9d. The ability of the chromionophore (or fluorionophore) to sense the true concentration of the specific ion is modulated by the amount of binding of the biological recognition molecule for its binding partner. The mechanism for this modulation of chromionophore (or fluorionophore) response to the binding between the biological recognition molecule and its binding partner can be direct or indirect as follows:
Direct modulation: The chromionophore (or fluorionophore) is attached chemically to a molecule having an affinity for the biological recognition molecule. Such a molecule is hereafter referred to as a xe2x80x9cchromaffinophore (or fluoraffinophore)xe2x80x9d. When the recognition molecule binds to the chromaffinophore (or fluoraffinophore), the chromionophore (or fluorionophore) portion is obstructed by the relatively large biological recognition molecule. This alters the chromaffinophore (or fluoraffinophore)""s ability to interact chemically with the ion to which it specifically responds.
Indirect modulation: The chromionophore (or fluorionophore) exists in an ion exchange context with a second ionophore which binds a second ion and transports it into a lipophilic environment such as a membrane. Charge neutrality must be maintained within the membrane. To achieve this, the membrane also contains a limited concentration of a lipophilic ion of opposite charge to that of the ions which are transported by the ionophore and chromionophore (or fluorionophore). Both the chromionophore (or fluorionophore) and the other ionophore compete for charge neutralization by this counter-ion in carrying their respective ions into the membrane. In the case of indirect modulation, the ionophore is attached chemically to a molecule having an affinity for the biological recognition molecule. Such a molecule is hereafter referred to as an xe2x80x9caffinophorexe2x80x9d. The binding of the recognition molecule to the affinophore alters its ability to transport its specific ion into the membrane. This makes more counter-ion available to the chromionophore (or fluorionophore) thereby permitting increased transport of its ion into the membrane and causing a shift in the ion concentration at which a color or fluorescence change occurs in the chromionophore (or fluorionophore).
Thus the test strip may be comprised in two possible combinations of components, the first combination being:
1a) an absorbent matrix through which the liquid containing the analyte to be measured is carried to a membrane.
2a) membrane affixed to and/or in contact with the matrix, said membrane containing
a) certain lipophilic ions, each of which has a certain electrical charge;
b) a chromionophore (or fluorionophore) which is also adapted to carry a first ionic species into said membrane where said ionic species will neutralize a portion of the certain electrical charge of a portion of said lipophilic ions, and which will change color (or fluorescence) when it carries an ion into the membrane.
c) A plurality of affinophores, each of which is adapted to carry into said membrane a second ionic species which after entering the membrane will neutralize the certain electrical charge of a portion of said lipophilic ions, and which possesses a binding affinity for a recognition molecule which binds to the substance being measured.
3a) a plurality of recognition molecules which bind both to the substance to be measured and to the affinophore, said recognition molecule being present on the absorbent matrix and/or added to or already present in the solution containing the analyte.
4a) a source of ions which will be transported by the chromionophores (or fluorionophores) and affinophores, said ions being contained in the absorbent matrix and/or added to or already present in the solution matrix, the concentration of said ions available to the membrane being either controlled (e.g. buffered) or in excess.
The second combination of components comprising the test strips is
1b) an absorbent matrix through which the liquid containing the analyte to be measured is carried to a membrane.
2b) membrane affixed to and/or in contact with the matrix, said membrane containing
a) certain lipophilic ions, each of which has a certain electrical charge;
b) a plurality of chromaffinophores (or fluoraffinophores) each of which is adapted to carry an ion or ions to said membrane, and which will change color (or fluorescence) when it carries an ion into the membrane, and which possess a binding affinity for a recognition molecule which binds to the analyte being measured.
3b) a plurality of recognition molecules which bind to both the analyte to be measured and to the chromaffinophore (or fluoraffinophore), said recognition molecules being present on the absorbent matrix and/or added to or already present in the solution being measured.
4b) a source of ions which will be transported by the chromaffinophores (or fluoraffinophores) said ions being contained in the absorbent matrix and/or added to or already present in the solution matrix, the concentration of said ions available to the membrane being either controlled (e.g. buffered) or in excess.
Either of the above embodiments may be modified to incorporate the placement of a chemical marker at some point on the strip, said chemical marker providing a visible change resulting from exposure of the marker to the liquid which carries the sample. This marker serves to alert the user that the solvent front carrying the sample has reached the point at which the strip should be read.
According to a second aspect of the present invention, a method is provided to determine the concentration of a certain analyte occurring within a solution. When used with the embodiment of the apparatus which does not possess the ionic species impregnated into the absorbent matrix, the method comprises the following steps:
1) adding a specified quantity of recognition molecules and/or of ionic species and/or buffering compounds to a specified volume of sample.
2) Mixing the sample
3) Waiting a prescribed period of time greater than one (1) second and less than one (1) hour.
4) Placing a test strip of the type described in the first aspect of invention into a volume of a sample which is to be tested for a time sufficient to allow the sample to permeate the absorbent matrix and/or to chromatograph into the matrix, so as to bring the sample and recognition molecule into contact with the membrane, thereby allowing recognition molecules which are not bound to the analyte in the sample to bind to the affinophore or chromaffinophore (or fluoraffinophore) in the strip molecules, thereby affecting the color or the membrane in a manner related to the concentration of said analyte within said medium.
5) Stopping the flow of the sample into absorbant matrix either by removing the strip from the sample or by controlling the volume of sample so that the supply is exhausted.
6) Reading the test strip with the help of a calibrated scale provided with the test strip kit.
Additionally, according to a second aspect of the present invention, a second method is also provided to determine the concentration of a certain analyte occurring within a solution. This method will be used with the embodiment of the apparatus which possesses both the ionic species and the recognition molecules impregnated into the absorbent matrix. The method comprises the steps:
1) placing a test strip of the type described in the first aspect of the present invention into a volume of a sample which is to be tested for a time sufficient to allow the sample to permeate the absorbent matrix and/or to chromatograph into the matrix, so as to bring the sample and recognition molecule into contact with the membrane, thereby allowing recognition molecules which are not bound to the analyte in the sample to bind to the affinophore or chromaffinophore (or fluoraffinophore) in the strip molecules which are not bound to the analyte in the sample to bind to the affinophore or chromaffinophore (or fluoraffinophore) in the strip molecules, thereby affecting the color or the membrane in a manner related to the concentration of said analyte within said medium.
2) Stopping the flow of the sample into absorbant matrix either by removing the strip from the sample or by controlling the volume of sample so that the supply is exhausted.
3) Reading the test strip with the help of a calibrated scale provided with the test strip kit.
Third and fourth embodiments of the second aspect of the invention result in the case of strips which incorporate the placement of a chemical marker at some point on the strip, said chemical marker providing a visible change resulting from exposure of the marker to the liquid which carries the sample. This marker (hereafter referred to as xe2x80x9cthe end point markerxe2x80x9d) serves to alert the user that the solvent front carrying the sample has reached the point at which the strip should be read. For these strips, step 1 of the procedures becomes:
1) placing a test strip of the type described in the first aspect of the present invention into a volume of a sample which is to be tested for a time sufficient to allow the sample to permeate the absorbent matrix and/or to chromatograph into the matrix, so as to bring the sample and recognition molecule into contact with the membrane, thereby allowing recognition molecules which are not bound to the analyte in the sample to bind to the affinophore or chromaffinophore (or fluoraffinophore) in the strip molecules which are not bound to the analyte in the sample to bind to the affinophore or chromaffinophore (or fluoraffinophore) in the strip molecules, thereby affecting the color or the membrane in a manner related to the concentration of said analyte within said medium; said sufficient time being defined by a change in the end point marker.
Further objects, features, and advantages of the present invention will become apparent from a consideration of the following description and the appended claims when taken in conjunction with the accompanying drawings.